Salmonella enterica serovar Typhi (S. Typhi) is a bacterial pathogen that is well-known for causing the acute illness called typhoid fever, as well as for its ability to remain in carriers without causing symptoms and its resistance to multiple antibiotics. Vi capsular polysaccharides and other virulence factors are critical to S. Typhi's pathogenicity. The Vi capsule, the topic of this paper, acts as a shield against the host's innate immune responses, and S. Typhi without a capsule is generally considered to be less harmful or less potent than regular S. Typhi strains. On the other hand, Vi is the only antigen found in typhoid subunit vaccines that targets S. Typhi and is also utilized in one of the diagnostic tests for typhoid fever.

Our study was motivated by the need to understand whether there are Vi capsule variants of S. Typhi in circulation. Our study suggests that Vi capsule variants account for at least 20% of the S. Typhi strains currently in circulation. Our study combines bioinformatics, functional characterizations, and epidemiological analyses to examine a selection of clinical missense mutations.

Our recently assembled three-stage bioinformatics pipeline revealed that two specific enzymes involved in Vi capsule synthesis, TviE and TviD, concentrated the majority of missense mutations. To investigate the potential differences in Vi capsule, we chose to focus on 21 clinical missense mutations. Specifically, we wanted to see if variants of S. Typhi that had a single amino acid point mutation in either TviE or TviD displayed any distinct characteristics. By utilizing molecular and biochemical methods, we have made an intriguing finding: each of these specific point mutations leads to a distinct variant form of Vi. In comparison to the wild-type Vi, these variations show differences in amount, length, and/or acetylation. We categorized these mutations into three groups: hypo, hyper, and intermediate Vi capsule variants. Our study indicates that one of these mutations can cause changes in Vi capsule, likely due to the modified actions of TviE and TviD enzymes, such as polymerization or acetylation.

Hypo Vi capsules, such as S. Typhi with tviE K266N or tviD C127R, have shorter Vi (their exact glycan structures are to be unveiled) and are primarily found in Africa. Surprisingly, the hypo Vi capsule variants, despite their short Vi, also possess the ability to successfully evade the host's innate immune defense mechanisms, similar to S. Typhi with the wild-type Vi capsule. Our research findings suggest that the hypo Vi capsule has a significant advantage in terms of its enhanced ability to infect host cells. It was clear from both cell culture and mouse infection studies that the infectivity of hypo Vi capsule S. Typhi was significantly increased compared to wild-type S. Typhi.

Hyper Vi capsules (their exact glycan structures are to be unveiled), such as S. Typhi with tviE C137R, tviE P263S, tviE A462V, or tviD R508H, have been present in human populations for many years. Various strains of S. Typhi, including the hyper Vi capsule variants, are currently present worldwide. Hyper Vi capsules have been found to have a strong impact on virulence, as shown by the notable rates of illness and death observed in infected mice, as well as their superb ability to colonize the gallbladder. Both bacteria-associated and shed Vi are linked to a higher level of virulence in the hyper Vi capsule variants. The hyper Vi capsule on bacteria plays a critical role in evading neutrophil-mediated phagocytosis. Furthermore, the shed Vi contributes to the heightened infection of co-infected bacteria, such as S. Typhi with the wild-type Vi capsule. It is alarming that all instances of a specific hyper Vi capsule variant have demonstrated resistance to ciprofloxacin, a commonly used treatment for typhoid patients in clinical settings.

The intermediate Vi capsule group displays a phenotype that lies between the WT and hyper Vi capsule variants. Interestingly, several of the missense mutations in this group share similarities with the hyper Vi capsule variants.

Overall, our study raises awareness of the existence of many Vi capsule types of S. Typhi, both within and outside the scientific community. We have only begun to uncover Vi capsule variations, and more research will reveal numerous unknowns in this realm. This study lays the framework for future research on S. Typhi capsule variants and provides helpful information on how to combat S. Typhi bacteria carrying these capsule variants.